Cryopreservation is a procedure for preparation of a suspension of cells, or a group of cells such as an embryo, for storage. The procedure normally incorporates adding cryoprotectants to the cells to be preserved, cooling of the suspended cells, long-term storage of the cell suspension at temperatures below about -80.degree. C., warming of the cells to normal cell temperatures, and removal of cryoprotectant from the cells. Cryopreservation of sperm or other cells from common mammals is a deceptively simple-appearing process which succeeds despite certain serious obstacles. This success depends on the use of one or more cryoprotectants in the context of certain procedural parameters.
Overall cryopreservation procedures generally thus include: preparation of a suspension of cells for low-temperature storage by incorporation of cryoprotectants, and placement of individual units into vials or "straws"; cooling (sometimes called "freezing") at an appropriate rate; long-term storage of the suspension of cells at a temperature lower than -80.degree. C. and often between -180.degree. C. and -196.degree. C.; distribution at low temperature to intermediaries or users; warming (sometimes called "thawing") at an appropriate rate to the normal cellular temperature; and controlled removal of cryoprotectant plus any other medium or other adjustments needed to render the cells ready for in vivo use. The goal is not just to keep cells alive (viable), but to optimize retention of all cellular attributes such as normal life span, oxygen-carrying potential (especially in the case of erythrocytes) and fertilizing potential (in the case of spermatozoa or oocytes), for which the cells are being preserved in the first place.
Notwithstanding cell type, species of origin or the various protocols used, prior art cryopreservation protocols traditionally result in about 30% mortality (or worse) of cells being preserved. Many cells traditionally did not survive the cooling and rewarming, and those which did suffered further damage during the removal of the intracellular cryoprotectant. Damage can result from any or all of improper rates of temperature changes during cooling and rewarming; formation of ice crystals; reduction in temperature per se; toxicity due to high concentrations of solutes within and around the cells; the nature and concentration of the cryoprotectant(s) used; rates of addition and removal of cryoprotectants from within the cells; and other lesser known but empirically evident factors.
Depending on the number of cells required for a functional "unit" after thawing, cells are traditionally packaged in individual "units" using glass ampules, plastic vials, plastic straws, or appropriately sized plastic bags. These packages all require removal of the cell suspension from the primary container before slow removal of cryoprotectant. Slow removal of cryoprotectant conventionally is achieved by dilution followed by centrifugation to re-concentrate the cell suspension or by washing cells via counter-flow centrifugation. Alternatively, technology disclosed in U.S. Pat. Nos. 5,026,342 and 5,261,870, incorporated herein by reference, allow removal of cryoprotectant from a primary container while cells of that individual unit remain therein; such primary containers are formed with walls or membranes having plugged pores which can be opened at a predetermined time to retain cells within the container but to allow exchange of fluid and molecules through the pores.
It is a common practice in the cryopreservation of cells: (1) to seal the primary container (such as a vial or straw) after it is filled with cells and before freezing; (2) to cool (sometimes called freezing) individual units at an appropriate rate to -196.degree. C.; (3) to group the primary containers in storage "boxes" or "goblets and canes" after which they are held at low storage temperatures such as -196.degree. C.; and (3) removing primary containers from this storage box or cane before warming (sometimes called thawing) the contents of the primary container by procedures known to those skilled in the art. U.S. Pat. No. 3,743,104 discloses a system in which straws containing semen are sealed by procedures known in the art and then placed into a "special cane" in which the metal has been formed to hold two groups of 4-10 straws during subsequent freezing and storage, with the straws being removed individually from the special cane prior to thawing. Use of a special cane in this way is more labor intensive than is optimal.
Conventional procedures for preservation of many types of cells have involved controlled cooling from room temperature of 4.degree. C. to -80 or -100.degree. C., followed by plunging the containers of cells into liquid nitrogen at -196.degree. C. or storage in vapor above liquid nitrogen. This reflects recognition that there are complex interactions among rate of cooling, concentrations of cryoprotectants and other solutes, temperature or spontaneous or planned "seeding," and other factors. It is equally well known that there is an interaction between cooling and warming rates but there has been no practical method to achieve a defined warming rate from at or below -40.degree. C. to 4.degree. C. Warming rate has been controlled passively on the basis of the surrounding medium (i.e., ambient air, ice water, 37.degree. C. water) rather than what is desirable from the standpoint of optimization of cellular attributes. Also, when water is used as a warming liquid, there is concern that water might leak into the primary package containing the cells and kill or seriously damage them.
A need thus remains for a mechanical means to arrange, store and transport individual units of cells intended for low temperature storage, which means provides positioning for the units from the time the initial cellular suspension is charged therein throughout many subsequent steps including but not limited to cooling and storage.